VacA and cagA genotypes of Helicobacter pylori isolated from raw meat in Isfahan province, Iran.

Foods with animal origins play a substantial role in the transmission of Helicobacter pylori. The present investigation was carried out to study the vacA and cagA genotypes status of H. pylori isolated from various types of meat samples. Two hundred and twenty meat samples were collected and cultured. H. pylori-positive strains were analyzed for the presence of vacA and cagA genotypes. Eleven out of 220 (5.00%) samples were positive for H. pylori. Findings were confirmed by nested PCR. Prevalence of H. pylori in the meat samples of slaughterhouses and butcheries were 72.20% and 27.70%, respectively. The most commonly detected genotypes in the meat samples of slaughterhouses and butcheries were vacAm1a (66.66%) and vacA s1a (37.50%), respectively. The S1am1a was the most commonly detected genotype. Meat sampled from butcheries had the higher prevalence of H. pylori and its genotypes than those of slaughterhouses (p < 0.05). Results showed that meat samples could be the potential sources of virulent strains of H. pylori. Application of sanitary measures in the storage, transportation and sale of meat is essential for reducing the levels of H. pylori cross contamination.


Introduction
Meat as the most valuable livestock product is composed of protein and amino acids, minerals, fats and fatty acids, vitamins and other bioactive components which are beneficial for human health. From the nutritional point of view, meat's importance is derived from its high quality protein, containing all essential amino acids and highly bioavailable minerals and vitamins. In addition, presence of essential fatty acids multiplies the nutritional value of this food. 1,2 In keeping with this, meat can convert into the luscious, energetic and graceful products like sausage and hamburger. Due to the high consumption rate of meat and meat products in the world, observation of sanitary conditions is critical in their production and distribution.
Helicobacter pylori is a microaerophilic gram negative and spiral shaped bacterium which its main reservoir is humans, predominantly the human stomach. It colonizes most of the population, making it one of the most provocative bacteria in the world. It is the most suspected cause of peptic ulcer disease, duodenal ulcer, type B gastritis, gastric adenocarcinoma and mucosa-associated lymphoid tissue and lymphoma. 3 It has been estimated that 17 to 86.00% of hospitalized patients with peptic ulcers were infected with H. pylori. [3][4][5] The main routes of H. pylori infection have not been identified yet 2 . However, it is prospective that H. pylori infection occurs throughout childhood or adolescence both in developed and developing countries. 5 The main routes for transmission of H. pylori are person to person contacts especially fecal-oral or oral-oral. 6 The role of foods in the transmission of H. pylori is still unknown but there were several novel investigations focused on the identification of this bacterium in various types of food samples. [6][7][8][9][10] Suitable conditions like appropriate pH, moisture and temperature cause H. pylori to be easily survive in various types of foods including vegetable, salad, milk and meat. 8,10 To appraise the pathogenicity of H. pylori, evaluation of latent virulence factors and genotypes is essential. The most commonly important virulence factors among H. pylori strains of different clinical outcomes of human and animal are the vacuolating cytotoxin (vacA) and cytotoxin associated gene (cag). 8,11,12 These genes usually induce adhesion and invasion to the gastric epithelial cells. 13,14 The vacA belongs to the group of genes with variable genotypes or structures. This gene is associated with injury to epithelial cells. The vacA gene is polymorphic, comprising variable signal regions (type s1 or s2) and midregions (type m1 or m2). The s1 type is further subtyped into s1a, s1b and s1c subtypes and the m1 into m1a and m1b subtypes. The mosaic combination of s and m-region allelic types determines the particular cytotoxin and consequently, the pathogenicity of the bacterium. [13][14][15] The Cag pathogenicity island (PAI) has been shown to be involved in persuading inflammation, ulceration and carcinogenesis. 14 The cagA gene has been detected in the specimens taken from the severe cases of peptic ulcer. [10][11][12][13][14] Genotyping using these virulence markers is considered as one of the best approaches for study of correlations between H. pylori isolates from different samples.
Data on the epidemiology and transmission of H. pylori are extremely significant to prevent its distribution and identify high-risk populations, especially in areas that have high rates of gastritis, peptic ulcers and gastric cancer like Iran. [8][9][10][11][12]15 Considering the unclear epidemiological aspects of H. pylori in meat and meat products and due to the high prevalence of H. pylori all around the world, the present investigation was carried out in order to study the exact status of vacA and cagA genotypes of H. pylori isolated from various types of meat and meat products.

Materials and Methods
Sample collection. From July to September 2015, overall 220 meat samples including beef (n = 80), mutton (n = 70) and chevon (n = 70) were collected from slaughterhouses and butcheries of various parts of Isfahan province, Iran. Samples (100 mg, in sterile glass containers) were transferred to laboratory at 4 ˚C and then refrigerated in plastic bags; information about dates of production and of assigned shelf lives was not presented.
DNA extraction and nested PCR amplification. Typical colonies of H. pylori were further identified using nested-PCR method. Genomic DNA was extracted from typical colonies using a DNA extraction kit for cells and tissues (Fermentas, St. Leon-Rot, Germany) according to the manufacturer's instructions and its density was assessed by optic densitometry. The first and second steps of PCR was performed based on the method described previously. 16 Genotyping of vacA and cagA genes of H. pylori. Presence of the vacA and cagA alleles were determined using PCR technique. 17,18 All PCR reactions were done using the programmable thermal cycler (Eppendorf Co., Hamburg, Germany). All runs included one negative DNA control consisting of PCR grade water and two or more positive controls (26695, J99, SS1, Tx30, 88-23 and 84-183). A PCR method used for amplification of various genotypes of vacA was done with a total volume of 50 µL including 2 mM Mgcl2, 1 µM of forward primer, 1 µM of reverse primer, 5 µL PCR buffer 10X, 200 µM dNTP (Fermentas), 1 U Taq DNA polymerase (Fermentas) and 2.50 µL DNA template. The DNA was then amplified by 32 successive cycles of denaturation at 95 ˚C for 45 sec, primer annealing at 64 ˚C for 50 sec and DNA chain extension at 72 ˚C for 70 sec. Similar PCR reaction was done for the cagA genotype. Amplified DNA of cagA PCR method was then amplified by 32 successive cycles of denaturation at 95 ˚C for 60 sec, primer annealing at 56 ˚C for 60 sec and DNA chain extension at 72 ˚C for 60 sec.
Gel electrophoresis. The PCR amplification products (10 μL) were subjected to electrophoresis in a 2% agarose gel in 1X TBE buffer at 80 V for 30 min, stained with SYBR Green and images were obtained in a UVI doc gel documentation systems (Uvitec Limited, Cambridge, UK). The PCR products were identified by 100 bp DNA size marker (Fermentas).
Statistical analysis. Data were transferred to Microsoft Excel spreadsheet (version 15; Microsoft Corp., Redmond, USA) for analysis. Using statistical software (version 16; SPSS Inc., Chicago, USA), Chi-square test and Fisher's exact two-tailed test analysis were performed and differences were considered significant at values of p < 0.05. Distribution of H. pylori genotypes isolated from meat samples was statistically analyzed. Table 1 represents the total prevalence of H. pylori in various types of meat samples. Eleven out of 220 (5.00%) meat samples were positive for the H. pylori in the culturebased method. The results of culture method were confirmed by nested PCR technique. Total prevalence of H. pylori in meat of slaughterhouses and butcheries were 2.72% and 7.27%, respectively.

Results
Totally, mutton from butcheries (11.42%), mutton from slaughterhouses (5.71%) and chevon from butcheries (5.71%) had the highest prevalence of H. pylori. The meat samples taken from butcheries had the higher prevalence of H. pylori than those of slaughterhouses (p < 0.05). Table 2 shows the distribution of various genotypes in H. pylori strains of meat and meat products. The most commonly detected genotypes in the meat samples taken from butcheries and those of slaughterhouses were vacA m1a (66.66%) and vacA s1a (37.50%), respectively. The H. pylori strains of butcher's meat samples harbored the higher prevalence of all studied genotypes than those of slaughterhouses (p < 0.05). Table 3 reveals the distribution of combined genotypes in the H. pylori strains of meat and meat products. The S1am1a was the most commonly detected genotype in the H. pylori strains of all studied samples. The most commonly detected genotype in the H. pylori strains of slaughter-houses and butcheries were s1am1a, too.

Discussion
The results of the present investigation revealed that the meat samples of slaughterhouses and butcheries were contaminated with virulent strains of H. pylori. We found that the meat samples of butcheries had the higher prevalence of H. pylori. However, the main reason for this finding is uncertain but it seems that cross contaminations of meat samples after slaughter are maybe the main factor for the higher prevalence of H. pylori in butcheries. Increase in the prevalence of human-based genotypes in the meat samples from butcheries is another reason for the possibility of cross contamination. In fact, meat washing, storing and transporting are the main stages which may increase the prevalence of H. pylori contamination. Reports showed that H. pylori can survive in water. 19,20 In addition, the number of investigations which were focused on the isolation of H. pylori from the water samples are significant. 19,20 The lack of drinking water for washing of animal carcass and using unsanitary water in slaughterhouses is one of the main factors for presence of H. pylori in meat samples. Transmission of H. pylori strains form the hands of infected staff and also butcheries is another reason for high prevalence of H. pylori. Possibility for contamination of animal carcasses in various stages of slaughter with intestinal contents, blood, feces, wool of animal and also slaughterhouse equipment such as knives, saws and hands of the meat inspector's and sometimes butcheries attended for meat purchasing can be considered among other causes of meat contamination. The presence of exotic animals such as cats and birds in the line of slaughter is another factor which should not be ignored. To our best knowledge, this is the first prevalence report of isolation and genotyping of H. pylori in various types of raw meat samples in the world. Our results revealed that 2.72% of meat samples of slaughterhouses and 7.27% of meat samples of butcheries were contaminated with H. pylori strains. In comparison with other researches which were conducted on food staffs, the prevalence of H. pylori in our study was lower than dairy products (19.20%), 8 vegetable and salad (10.86%), 9 vegetables (13.68%), 10 restaurant salad (14.00%) 10 and milk (12.50%). 21 As far as we know, the prevalence found in this study is the lowest report for H. pylori in various types of food stuffs in the world.
Several studies have been focused on the detection of H. pylori in foods with animal origins. [23][24][25][26] An Italian investigation 24 showed that more than 25.00% of foods with animal origin were contaminated with H. pylori. In a study conducted in Japan, 72.20% of various types of animal-based foods were positive for H. pylori. 22 Despite other foods with animal origins, [22][23][24][25] prevalence of H. pylori in cattle, sheep and goat meat samples of our study was considerable. Prevalence of bacterium in Greek 24 and USA 25 was 20.00% and 60.00%, respectively, which was higher than our results in Iran. In a study by Mhaskar et  The second part of our investigation was focused on the vacA and cagA genotype status of H. pylori isolates of meat samples. Our results showed that cagA, vacA s1a, vacA m1a, vacA s2 and vacA m2 were the most commonly detected genotypes. In addition, s1am1a, s1am2, s2m1a and s2m2 were the most commonly detected combined genotypes. Unfortunately, the number of studies which were focused on the genotyping of H. pylori in food stuffs is low. A recent Iranian investigation conducted by Yahaghi et al. showed that from 380 vegetable and 50 salad samples, 52 (13.68%) and 7 (14.00%) were positive for H. pylori, respectively. 10 The most commonly detected genotypes in their investigation were oipA (86.44%), cagA (57.62), vacA s1a (37.28%) and vacA m1a (30.50%). Their results showed that vacA s1c had the lowest prevalence which was similar to our findings. Mousavi et al. showed that the most commonly detected genes in the H. pylori strains recovered from milk and dairy products were cagA (76.60%) and vacA (75.00%) which was similar to our findings. 8 Hemmatinezhad et al. reported that 13.45% of ready to eat food samples were positive for H. pylori. Meatbased ready to eat food samples were the most contaminated. 27 They revealed that the most commonly detected genotypes were vacA s1a (78.37%), vacA m2 (75.67%), vacA m1a (51.35%) and cagA (41.89%) which was similar to our findings. They showed that s1am2 (70.27%), s1am1a (39.18%) and m1am2 (31.08%) were the most commonly detected combined genotypes which was similar to our investigation. In a study which was conducted by Saeidi and Sheikhshahrokh 820 meat and milk samples were studied for presence of H. pylori and vacA genotypes. 28 They showed that the prevalence of H. pylori in meat samples was 26.25% which was higher than our results. Total prevalence of H. pylori in cow, sheep, goat, buffalo and camel meat samples was 25.00%, 37.00%, 22.00%, 28.00% and 14.00%, respectively. S1a and m1a were the most commonly detected genotypes. The most commonly detected combined genotypes were m1as1a (68.52%), m1as1b (60.40%), m1bs1b (55.83%) and m1bs1a (53.29%) which was similar to our findings.
The CagA, vacA s1a, m1a and m2 were the most commonly detected genotypes in the clinical food samples, 8,10,27,28 those of animal sources 8,21 and from human beings. [29][30][31]  Close association of vacA and cagA genotypes with production of interleukin-8 and cytotoxin, gastric epithelial cells adhesion, inflammatory effects, vacuolization and apoptosis in gastric epithelial cells has been observed previously. 32,33 Since H. pylori isolates in our study harbored vacA and cagA genotypes, consumption of meat contaminated with virulent strains may provoke duodenal ulceration, gastric mucosal atrophy and gastric cancer.
In conclusion, Iranian meat samples harbored H. pylori strains with high prevalence of vacA and cagA genotypes. High prevalence of H. pylori in our samples suggests that contaminated meat samples maybe the sources of bacteria and can transmit to the human population. Diversity of H. pylori genotypes between various types of meat samples showed that there might be various sources of contamination. The most important finding of this study is that the meat samples of our research harbored virulent strains of H. pylori. A robust health protocol on hygiene of slaughterhouses and the butcheries can reduce the risk of transmission of H. pylori from meat and its products to human.